The recognition potential (RP) is an early visually evoked response (~250 ms) whose magnitude is sensitive to object recognizability and related factors. The RP is often measured when objects are embedded in a rapid stream of masking stimuli (the RSS paradigm), especially in reading research. The idea is that RSS provides greater stimulus-dependent variations in RP, compared to the corresponding variations without RSS. However, this idea has never been subject to systematic evaluation. We directly test whether RSS can enhance 2 types of RP stimulus selectivity, by measuring the RP in conditions that only differ in the presence or absence of a masking stream and in the type of stimulus shown. We measure the effect of image inversion on RP for Chinese characters (experiment 1); the effect of orthographical correctness on RP for Chinese characters (experiment 3); and as a control study, the effect of image inversion on the N170 response to faces (experiment 2). To ensure a fair comparison, the earliest negative deflections (RP/N170) measured with and without RSS should at least have similar channel ranges, and topographical distributions of both amplitude and selectivity. Our first set of results clearly supports this. Our main results clearly show an increase in stimulus-selectivity for RSS over non-RSS in both of the RP (Chinese character) experiments, but no such enhancement in the N170 (face) experiment. This provides incentive for investigations into the underlying mechanisms of selectivity enhancement. Our findings may also help to explain contradictory findings between RP/N170 studies that differ only in the use of noise masks, which is sometimes treated as trivial detail in papers that do not reference the RP/RSS literature. The rapid-stream-stimulation procedure (RSS) is a paradigm for stimulus presentation that is thought to enhance early electrophysiological responses to recognizable stimuli. In this study, we test whether RSS increases the recognition potential , and whether it also affects selectivity measured with other stimuli (the N170 response to faces in this study).The recognition potential (RP) is an electrical brain response peaking around 200 to 250ms, obtained when subjects view recognizable images, such as words –3 or picThe N170 is an ERP component with a negative deflection that is evoked most strongly by familiar objects such as faces, words and cars . As desIn almost all of the RP studies we have cited, the rapid-stream-stimulation procedure (RSS) is used to present stimuli, because it is thought enhance the degree of RP selectivity between perceptually and semantically different stimulus categories that vary in recognizability. However, there is no direct evidence that RSS enhances RP selectivity in the types of designs that are used in the vast majority of studies. This paper describes 2 experiments designed to provide a direct test of RSS-enhancement of RP selectivity to linguistic stimuli, and 1 additional study that examines the effect of RSS on the selectivity of the N170 response to faces.The RSS procedure originates with Rudell , who devRudell himself devised the RSS based partly on an older paper from 1991  that sugSubsequent researchers have come up with additional reasons why RSS should provide a cleaner signal of RP –22, but Aside from , there iTherefore, we compare RSS and non-RSS RP’s in 2 different types of object-processing experiment most appropriate for eliciting a recognizability response in Chinese subjects (the available subject pool at our institute in China). And unlike Dien et al. , we use At this point, we’d like to emphasize that the goal of this study is to determine if RSS-enhancement of RP or N170 exists, and not to establish why it might exist. Nonetheless, possible mechanisms for RSS-enhancement should be briefly mentioned here to underscore the theoretical plausibility of RSS-enhancement. The most commonly provided explanation for why RSS might enhance RP requires a distinction between RP and earlier, purely visually-evoked components that might interfere with RP measurement. As Martín-Loeches  wrote: “Our experiments were conducted in China using Chinese subjects. To ensure recognizability, our stimuli were either Chinese characters (experiments 1 and 3), or photographs of real Chinese faces (experiment 2). To date, the only RP experiments using Chinese characters and subjects derive from two studies –13. ExpeAs an additional study, experiment 2 contrasts N170 responses to upright and inverted Chinese faces. The focus on orientation selectivity of N170 in this experiment is motivated by the same reason provided for the first experiment—to rule out low-level image properties as an explanation for N170 sensitivity to face orientation. Compared to upright faces, the N170 is found to be enhanced for inverted faces indicated by a longer latency and larger amplitude –26.Our cExperiment 3 is perhaps the most strongly motivated by the previous literature and has the greatest expectation for RSS enhancement of RP selectivity. Chinese characters are of unique interest in detailing the specific forms of linguistic processing that may underlie early evoked responses. The reason for this is the mostly arbitrary mapping between visual and phonological forms. This property of characters therefore allows for the investigation of visual form familiarity with minimal involvement of phonology. To that end, Lin et al.  measuredFor those unfamiliar with RP research, it is important to point out that an early evoked response to a recognizable linguistic stimulus (first negative deflection) is almost always labelled an RP regardless of whether RSS has been employed or not; and regardless of whether the same component might be called an N170 by other researchers who may be unaware of the RP literature e.g., –28). Thi. Thi28])For each of the 3 experiments, subjects performed both an RSS (masked) and non-RSS (unmasked) version of that experiment on separate occasions. The procedure used for our RSS and non-RSS paradigms were as closely matched as possible. In fact, the only difference in procedure is that each presentation of a masking stimulus used in the RSS paradigm is replaced with a blank screen in the N170 paradigm, of equal duration see . TherefoGiven previous results, we can be more specific about the direction of some of our expected effects. In experiment 1, we expect upright characters to evoke an RP of higher amplitude than that evoked by inverted characters –13. CritAs mentioned before, the RP measured in RSS is likely to occur in a later time window, due to the effects of the forward mask. If so, then this raises the possibility that the presence of noise, or some other aspect of the RSS procedure, has shifted the RP response to another electrode, another time-window, or both. Therefore, it is important that we establish that both types of RP—with and without RSS—share important aspects of topography and selectivity. It is also important that, in our evaluation of enhanced stimulus selectivity, we examine a range of likely electrodes. Therefore, the first half of our ERP results is a comparison of RP/N170 topography and timing between RSS and non-RSS paradigms.All participants  in all experiments were Chinese students who participated as paid volunteers. They were all right-handed, had normal or corrected-to-normal vision and had no neurological history. The experiments reported here, the informed consent, and all experimental protocols, was approved by the Institutional Review Board of Hangzhou Normal University. All methods were performed in accordance with the approved guidelines and regulations. Informed consent was obtained from all subjects. Each participant signed a written, informed consent form prior to the ERP experiment and was compensated for CNY50 per hour after the experiment.We selected 100 Chinese characters from the same database used in experiment 3 . The frequency of these characters ranged from 877.7/million to 2536/million . The stroke number of the characters ranged from 7 to 11 . The inverted characters were created by rotating the upright characters 180 degrees. The characters were grayscale (black on grey) and the visual angle was 4.4°x4.4°. The masks were created using the same method used in the lexicality experiment.Faces of neutral expression were taken from the CAS-PEAL-R1 face database . We selehttp://ccl.pku.edu.cn:8080/ccl_corpus/index.jsp?dir=xiandai) and they were all left-right compound characters. Each pseudo character was created by combining the left radical of one real character, with the right radical of another real character; thus creating a novel character whose radicals are each in their correct position . We looked through a Chinese dictionary (中华大字典) . On average, accuracy was lower for the RSS paradigm. The mean difference of proportion correct (RSS minus non-RSS) was -0.031 .A repeated-measures 2 (Paradigm) x 2 (Stimulus) ANOVA of reaction-time (RT) revealed significant main effects of paradigm  and stimulus , and an interaction . Pair-wise t-tests comparing RSS and non-RSS paradigms were performed separately for upright and inverted stimuli. This demonstrated a significant non-RSS speed advantage for both upright and inverted stimuli. For upright stimuli: (t(23) = 5.8, p = 5.9e-06), with a 95-percent confidence interval of  around a mean difference of 0.054 seconds. For inverted stimuli: (t(23) = 8.5, p = 1.5e-08), with a 95-percent confidence interval of  around a mean difference of 0.067 seconds. All t-tests significant with Bonferroni correction (criterion p = 0.05/2 = 0.025).Mean accuracies and RT are reported in Tables A repeated-measures 2 (Paradigm) x 2 (Stimulus) ANOVA of accuracy revealed no significant effects.A repeated-measures 2 (Paradigm) x 2 (Stimulus) ANOVA of reaction-time (RT) revealed significant main effects of paradigm , and stimulus , but no interaction. RSS was slower than non-RSS by a mean of 0.049 seconds . And inverted was slower than upright by a mean of 0.013 seconds .Mean accuracies and RT are reported in Tables A repeated-measures 2 (Paradigm) x 2 (Stimulus) ANOVA of accuracy revealed significant main effects of paradigm , and stimulus , but no interaction. The mean difference of proportion correct (RSS minus non-RSS) was -0.048 . Two post-hoc paired t-tests were done to compare stimuli. Difference of proportion correct  was significant: (t(47) = -2.8, p = 0.0073), with a 95-percent confidence interval of  around a mean difference of -0.086. Additionally, non-word minus pseudo proportion correct was also significant: (t(47) = 6.7, p = 2e-08), with a 95-percent confidence interval of  around a mean difference of 0.16. All t-tests significant with Bonferroni correction (criterion p = 0.05/2 = 0.025).A repeated-measures 2 (Paradigm) x 2 (Stimulus) ANOVA of RT revealed significant main effects of paradigm , and stimulus , and an interaction . Three post-hoc paired t-tests examined (RSS minus non-RSS) RT (seconds) separately for each stimulus type, and each was significant. Positive values of RT difference indicate that reactions were slower for the RSS paradigm compared to the non-RSS paradigm. Real words: (t(23) = 7, p = 3.5e-07), with a 95-percent confidence interval of  around a mean difference of 0.046. Pseudo words: (t(23) = 5, p = 5.2e-05), with a 95-percent confidence interval of  around a mean difference of 0.049. And non-words: The effect was significant (t(23) = 9.3, p = 2.8e-09), with a 95-percent confidence interval of  around a mean difference of 0.068. All t-tests significant with Bonferroni correction (criterion p = 0.05/3 = 0.0167).The results showed that responses in the non-RSS paradigm are significantly more accurate and rapid than in the RSS paradigm; except for accuracies measured in Experiment 2 (faces). Participants responded most accurately and rapidly for the false characters and least accurately and rapidly for the pseudo characters. Overall, our behavioral results demonstrate the effectiveness of our masks on perception, and the effectiveness of our stimulus manipulation on lexicality.In In the RP/RSS literature, a common way of identifying channels that best capture the RP component is to select channels (sometimes among a posterior subset) that give the most negative first deflection after stimulus onset , 23–24. Perhaps more important than raw topography and component magnitude is stimulus selectivity. There is a possibility that non-RSS and RSS paradigms have selectivities that reside in different channel regions. However, we can see if this channel-switching actually occurs by examining the scatterplots shown in Can the topography of the RP/N170, at the non-RSS latency, predict the topography at the RSS latency? For each subject, in each condition, of each experiment, we obtain the topography of the RP/N170 at non-RSS latency. Then we measure the inner-product between this non-RSS topography and every topography along that subject’s RSS time course. If non-RSS and RSS components are similar, then the maximal inner-product should occur at or around the RSS latency itself. Therefore, we expect differences between the expected RSS latency (based on inner-products with non-RSS topography) and the true RSS latency (based on the timing of the first negative deflection in RSS data) to be close to zero. Large temporal offsets may suggest that RSS and non-RSS are not comparable at the same channel. But another explanation is that the non-RSS topography is not unique within its own time course. Therefore, a baseline measurement of temporal offset should be considered: the difference between expected non-RSS latency and true non-RSS latency. Offsets for predicted RSS latency (between-paradigm inner-products) can only be as small as offsets for predicted non-RSS latency (within-paradigm inner-products). For all comparisons, we obtain ninety-five percent confidence intervals for the 20-percent trimmed mean of latency offsets, using a percentile bootstrap; a procedure with many advantages  . ResultsA repeated-measures 2 (Paradigm) x 2 (Stimulus) x 2 (Hemisphere) x 3 (Electrode) ANOVA revealed a significant main effect of electrode , a significant main effect of stimulus , a paradigm x stimulus interaction , a stimulus x electrode interaction , and a paradigm x stimulus x electrode interaction . See Following up on our 3-way interaction (paradigm x stimulus x electrode), we performed separate 2-way ANOVA (paradigm x stimulus) for each electrode group: O1/O2, TP9/TP10, and P7/P8. Those results led to a total of 6 post-hoc t-tests, so those tests were tested for significance against a criterion p (0.05 / 6 = 0.008), as per Bonferroni correction.For O1/O2, there was both a main effect of stimulus , and a paradigm x stimulus interaction . Pair-wise t-tests comparing upright and inverted characters were performed separately for non-RSS and RSS paradigms. This demonstrated a significant stimulus effect for RSS: (t(95) = 15, p = 5.1e-26), with a 95-percent confidence interval of  around a mean difference of 2.1. However, no stimulus effect was found for the non-RSS paradigm: (t(95) = 0.49, p = 0.63), with a 95-percent confidence interval of  around a mean difference of 0.061.The same result was found for TP9/TP10. A 2-way ANOVA (paradigm and stimulus) revealed a significant interaction ; and pairwise t-tests revealed significant stimulus effects for the RSS paradigm, but not for the non-RSS paradigm. The t-test for non-RSS paradigm gave (t(95) = -1.4, p = 0.16), with a 95-percent confidence interval of  around a mean difference of -0.19. While the t-test for RP gave (t(95) = 5.6, p = 2e-07), with a 95-percent confidence interval of  around a mean difference of 0.64.A slightly different result was obtained for P7/P8. The 2-way ANOVA (paradigm and stimulus) did reveal a significant interaction: . However, t-tests comparing stimuli performed separately for non-RSS and RSS, both revealed a significant effect of stimulus. The t-test for non-RSS gave (t(95) = -3.5, p = 0.00079); and the t-test for RSS gave (t(95) = 10, p = 3.8e-17). Nonetheless, the magnitude of the stimulus effect was noticeably larger for RSS than for non-RSS: the 95-percent confidence interval and mean difference was  and -0.52 for non-RSS, while it was  and 1.4 for RSS.A repeated-measures 2 (Paradigm) x 2 (Stimulus) x 2 (Hemisphere) x 3 (Electrode) ANOVA revealed a significant main effect of electrode , stimulus , hemisphere , and a stimulus x electrode interaction . See Following up on our 2-way interaction (stimulus x electrode), we performed separate t-tests (stimulus) for each electrode: O1/O2, TP9/TP10, and P7/P8. While the magnitude of stimulus effects varied across electrode, a significant effect was obtained for each. For O1/O2: (t(191) = -17, p = 5.2e-41), with a 95-percent confidence interval of  around a mean difference of -2.4. For TP9/TP10: (t(191) = -11, p = 8e-21), with a 95-percent confidence interval of  around a mean difference of -1.1. And for P7/P8: (t(191) = -16, p = 2e-16), with a 95-percent confidence interval of  around a mean difference of -2.4. All t-tests were significant with Bonferroni correction (criterion p = 0.05/3 = 0.0167).A repeated-measures 2 (Paradigm) x 2 (Stimulus) x 2 (Hemisphere) x 3 (Electrode) ANOVA revealed a significant main effect of stimulus , electrode , and a paradigm x stimulus interaction . See Following up on our 2-way interaction (paradigm x stimulus), we performed separate 1-way ANOVA  for each paradigm (non-RSS and RSS).While there was no significant effect of stimulus for the non-RSS paradigm, the RSS paradigm did reveal an effect: . Two pairwise t-tests were performed to evaluate the stimulus differences obtained in the RSS paradigm: real versus pseudo characters, and pseudo versus false. Real characters did not produce significantly different amplitudes from pseudo characters: (t(287) = 0.46, p = 0.65), with a 95-percent confidence interval of  around a mean difference of 0.023. However, pseudo and non-characters were significantly different: (t(287) = 8, p = 2.5e-14), with a 95-percent confidence interval of  around a mean difference of 0.46. We used Bonferroni correction (criterion p = 0.05/2 = 0.025).Please see the This study presents the first systematic and fair comparison of both RP and N170 selectivity between RSS and non-RSS paradigms. We confirm prior assumptions about RSS by demonstrating that the RSS paradigm can enhance the stimulus selectivity of RP component amplitude in 2 different experiments that use Chinese characters as stimuli. Our enhanced ‘orthography effect’ in particular (experiment 3), may help to resolve contradictory results by showing noise-masking may be necessary to even obtain a stimulus selectivity. These positive results with RP responses to Chinese characters contrast with our face study, which shows that RSS does not enhance the N170 selectivity to face orientation. Unlike the familiar Chinese characters and radicals that we use in experiments 1 and 3, our Chinese faces were unfamiliar to our participants. Therefore, our results with unfamiliar faces is consistent with the notion that RSS enhances selectivity to aspects of the stimuli that are distinctly recognizable, and richer with semantic association. Finally, we include a complementary body of results, including some novel analyses, which addresses the important question of whether N1 components measured with and without RSS are comparable in the first place. Our results show that, at least for the stimuli we use (faces and Chinese characters), the answer is yes.We took great care to ensure that the RP and N170 components we measured were similar enough between paradigms to be directly compared. First, we visualize grand-average topographies of RSS and non-RSS components, at their respective latencies, for each experiment and stimulus type. Second, we show separate histograms for RSS and non-RSS data, each showing the distribution of the most-negative first deflection across 10 posterior channels. Third, we use scatterplots to show how the stimulus-selectivity across channels changes between RSS and non-RSS paradigms. The second and third analyses help to determine if there is any ‘channel switching’ between RSS and non-RSS paradigms. We do not find any evidence of this. So, the final analysis is restricted to within-channel comparisons and focuses on the comparability of RSS and non-RSS timings. Indeed, RSS component latencies are well predicted by their topographical similarity to the non-RSS component. Importantly, these measures of topographical similarities (inner-products) are calculated separately for each subject and condition. Finally, as a complement to the preceding comparison between non-RSS and RSS components, our main result employs an ANOVA that includes both electrode and hemisphere as factors . RSS-enhancement suggests an interaction between stimulus  and paradigm (RSS versus non-RSS). If masking also alters the topography of stimulus selectivity then that should emerge as an additional interaction with electrode or hemisphere , and a specific pattern of post-hoc results suggesting that masking shifts selectivity to different electrodes, rather than simply amplifying selectivity at the same electrode(s). None of these analyses provided evidence that supports a difference of RP or N170 components between RSS and non-RSS paradigms.The presence of object-related amplitude differences in the RP and N170 are the focal points of many electrophysiological studies. Much less discussed, or even reported, are the strength of those effects. Given that the main topic of this paper is an enhancement of ERP effects, we were careful to include effect sizes. We consider these results to be critical for the evaluation of all our effects. It is also useful to consider our RSS-based character inversion-effect in light of the prevailing notion for what is a strong ERP effect, and what is typically obtained for N170 inversion effects. Using RSS, we obtain an average inversion-effect of 2.1  for electrodes O1/O2 and 1.4  for P7/P8. In comparison, typical N170 studies often report significant stimulus selectivities that are based on a 1 microvolt difference e.g. , 43); wh; wh43]);Our character ‘orthography effect’ , while clearly enhanced by the presence of a forward mask, is a weaker effect compared to the inversion effect. Using RSS, this effect gives a mean difference of 0.46 microvolts with a confidence interval of . In this case, forward-masking may in fact be a requirement for obtaining an ‘orthography effect’. We feel that further examination of the character ‘orthography effect’ would greatly benefit from considering the inclusion of a forward mask; and perhaps manipulating the properties of that mask to better understand the specific nature of the stimulus selectivity that is being inferred.We focused on the first negative deflection in this study (RP/N170) for 3 main reasons. First, our non-RSS conditions were replications of previous studies that focus on the first negative deflection. Second, almost all studies that employ RSS focus on the first negative deflection (RSS). And finally, other components like the P2 are difficult to isolate in many subjects; which is probably why there is such a strong focus on first negative deflections in the literature. However, the modest size of our ‘orthography effect’ also suggests that we might benefit from an examination of other components. Interestingly, Greater care needs to be taken when contrasting our N170 and RP results. As noted in the introduction, these components are often treated as different types of components. So, while both are the earliest negative deflection in response to recognizable objects, we adopt convention in labelling our character-evoked responses as RP and our face-evoked responses as N170. In our study however, another distinction may be of equal or greater importance. As our face stimuli were unfamiliar while our Chinese characters and radicals were familiar, recognizability was restricted to the category level in the former while it extended to exemplar level in the latter case. Therefore, the RSS enhancement of component selectivity may be restricted to stimuli that are familiar at an exemplar-specific level. However, while many studies show that RP  are modulated by exemplar-specific familiarity, there is little evidence that the N170 is modulated by face familiarity. We therefore, propose that RSS-enhancement of face selectivity might be found instead in the n250 component. Not only is this component modulated by face familiarity, it also peaks at around the same latency as the RP . Of courA different approach to thinking about the difference between our N170 and RP results is to consider their neural generators. Different classes of stimuli like faces and written script may recruit different regions or networks of regions in the brain, which may vary in how RSS affects their sensitivity. Ideally, information about the neural generators of N170 and RP would derive from studies that use high spatial-resolution techniques combined with EEG. Unfortunately, such studies are not numerous. For example, Dien et al  is the oIt is important to point out that the longer-term goal of this project is to investigate RSS as a tool for the enhancement and understanding of early component selectivity, in a very broad sense. We’ve demonstrated RSS-enhancement using 2 types of stimulus contrasts: upright versus inverted characters, and pseudo/real versus false characters. Those contrasts are important to vision and reading researchers for different reasons, but both may benefit from RSS-enhancement. This raises the question: What other types of early component selectivity can RSS enhance? And what, like the face-inversion effect, would remain unchanged by RSS? We feel that these empirical questions are of interest to the scores of other researchers who, like us, (1) may be uncertain about their ability to detect selectivity in their intrinsically noisy data; and (2) would like to understand the basis of this selectivity by identifying the factors that can modulate it. Of course, there are many ways to both obtain and analyze ERP data that might enhance the particular selectivity one is interested in. The RSS procedure is but one tool that can be used at the stage of stimulus presentation. Alternative forms of analysis  include the aforementioned linear modelling approach –39, compIn all of our experiments, the sequence of stimulus-events, and their timing, were almost exactly matched between RSS and non-RSS trials. This strongly suggests that the enhanced inversion-effect and orthography-effect found for Chinese characters was due to the presence of a stream of masking stimuli. Furthermore, we used the same procedure to create the masking stimuli in both of our Chinese character experiments (experiments 1 and 3). However, our face experiment (2) did use a different procedure to generate masking stimuli. This does raise the possibility that our face masking stimuli could have been better designed to illicit RSS enhancement of N170 face selectivity. Pu et al.  show thaUnlike the visibility/cognitive-load trade-off, the classic pre-empt explanation of RSS-enhancement takes into account the dynamic nature of RSS masking: masks and target-images are separated in time and this separation is easily perceived. Nonetheless, the effect of forward masking on the critical neural generators might be equivalent to the effect of simply degrading the target-image itself. Consistent with that possibility, RSS did reduce behavioral performance consistently across our experiments. To truly establish the importance of the dynamical aspect of the pre-empt explanation, one would need to model how ERP measured separately for mask and target images can be used to predict the response evoked by a sequence of mask and target. This level of explanation is currently lacking in the RSS/RP literature. On the other hand, an empirical verification of the visibility/cognitive-load trade-off would at least require confirmation of RSS-enhancement in conditions where the target-image is directly degraded and there is only a single evoked response isolated in time. This is easier to do than modeling ERP dynamics and it is something we are currently exploring. In the fMRI literature, there is some indirect support that masking-based enhancement can occur without RSS dynamics. Kay & Yeatman  measuredIn addition to the aforementioned projects, we believe that a fruitful way to study RSS-enhanced stimulus selectivity in the future would be to simply explore how masks may obscure the diagnostic features that are critical for the task and stimulus at hand. In this sense, the RSS procedure, together with a manipulation of mask characteristics, may lead to some insight into the information processing that underlies the generation of object-evoked potentials. With these results and insights in mind, we find great value in the continued investigation of RSS-enhancement. And, as the results of our orthography experiment suggest, greater care needs to be taken when comparing previous studies that differ in apparently trivial details like noise masking.S1 File(PDF)Click here for additional data file.


Urotensin-II (UT-II) and its receptor (UTR) are involved in the occurrence of different epithelial cancers. In particular, UTR was found overexpressed on colon, bladder, and prostate cancer cells. The conjugation of ligands, able to specifically bind receptors that are overexpressed on cancer cells, to liposome surface represents an efficient active targeting strategy to enhance selectivity and efficiency of drug delivery systems. The aim of this study was to develop liposomes conjugated with UT-II (LipoUT) for efficient targeting of cancer cells that overexpress UTR. The liposomes had a mean diameter between 150 nm and 160 nm with a narrow size distribution (PI ≤ 0.1) and a doxo encapsulation efficiency of 96%. Moreover, the conjugation of UT-II to liposomes weakly reduced the zeta potential. We evaluated UTR expression on prostate  and colon (WIDR and LoVo) cancer cells by FACS and western blotting analysis. UTR protein was expressed in all the tested cell lines; the level of expression was higher in WIDR, PC3, and LNCaP cells compared with LoVo and DU145. MTT cell viability assay showed that LipoUT-doxo was more active than Lipo-doxo on the growth inhibition of cells that overexpressed UTR  while in LoVo and DU145 cell lines, the activity was similar to or lower than that one of Lipo-doxo, respectively. Moreover, we found that cell uptake of Bodipy-labeled liposomes in PC3 and DU145 was higher for LipoUT than the not-armed counterparts but at higher extent in UTR overexpressing PC3 cells (about 2-fold higher), as evaluated by both confocal and FACS. In conclusion, the encapsulation of doxo in UT-II-targeted liposomes potentiated its delivery in UTR-overexpressing cells and could represent a new tool for the targeting of prostate and colon cancer. Urotensin-II (UT-II) has been described as the most potent vasoconstrictor, superior to other vasoactive molecules, such as endothelin-1, noradrenalin, and serotonin . In addi2000) were purchased from Lipoid GmbH . 23-(Dipyrrometheneboron difluoride)-24-norcholesterol (Bodipy-cholesterol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide-(polyethylene glycol)-2000]   were obtained from Avanti Polar Lipids . Doxorubicin hydrochloride (doxo), cholesterol (chol), sodium chloride,  (Hepes), ethylenediaminetetraacetic acid (EDTA), iron thiocyanate (FeSCN3), sodium citrate, potassium chloride, citric acid, and Sepharose CL-4B were purchased from Sigma Chemical Co. . All Fmoc-amino acids were purchased from GL Biochem  with their corresponding side chain protections for the solid-phase peptide synthesis (SPPS). Acetonitrile, methanol (HPLC degree), and chloroform (ACS grade) were obtained from Carlo Erba . RPMI, DMEM, and FBS were purchased from FlowLaboratories . Tissue culture plasticware was acquired from Becton Dickinson . Rabbit antisera raised against α-tubulin and UTR were purchased from Santa Cruz Biotechnology . UT-II peptide was synthetized and purified by the research group of Prof. Paolo Grieco .1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and   was synthesized by adopting the solid-phase peptide synthesis (SPPS) using the Fmoc/lsewhere . Upon cylsewhere , an addilsewhere . The pro2000 or DPPC/chol/DSPE-PEG2000-maleimide at a 65 : 30 : 5 molar ratio were prepared by hydration of a thin lipid film followed by extrusion. Briefly, lipids were dissolved in 1 mL of a mixture chloroform/methanol (2 : 1 v/v); the resulting solution was added to a 50 mL round-bottom flask, and the solvent was removed under reduced pressure by using a rotary evaporator . Then, the resulting lipid film was hydrated with 1 mL of citrate buffer at pH 4 , and the resulting suspension was gently mixed in the presence of glass beads until the lipid layer was removed from the glass wall; after that, the flask was left at 50°C for 2 h. The liposome suspension was then extruded at 50°C using a thermobarrel extruder system , passing repeatedly the suspension under nitrogen through polycarbonate membranes (5 times for each membrane) with decreasing pore sizes from 400 to 100 nm . The external buffer was removed by ultracentrifugation  at 80,000 rpm, at 4°C for 40 min, and the liposomes were resuspended with 1 mL of phosphate buffer at pH 7.4 . After preparation, liposomes were stored at 4°C. Each formulation was prepared in triplicate. For intracellular penetration studies, fluorescent-labeled liposomes containing 0.1% (w/w) of Bodipy-cholesterol were prepared similarly by adding the fluorescent cholesterol in the lipid mixture of DPPC/chol/DSPE-PEG2000 or DPPC/chol/DSPE-PEG2000-maleimide during the liposome preparation.Liposomes composed of DPPC/chol/DSPE-PEG2000-maleimide with gentle magnetic agitation at room temperature, overnight. The resulting liposomes, so-called LipoUT, were then chromatographed on a Sepharose CL-4B column in phosphate buffer at pH 7.4. All liposome preparations were stored at 4°C. The formulations were prepared in triplicate.Cys-UT-II  was conjugated with liposomes prepared with maleimide-modified polyethylene glycol. Briefly, the UT-II peptide (0.250 mM) was conjugated to liposomes containing DSPE-PEGby remote loading [Doxo was encapsulated into liposomes  loading . Briefly3) solution or iron thiocyanate solution (0.1 N) and CHCl3. The concentration of DPPC was obtained by measuring the absorbance at 485 nm into the organic layer with an ultraviolet-visible spectrophotometer . Quantification of DPPC was carried out by means of a calibration curve with standard DPPC samples.The concentration of lipids present in the suspensions after preparation was determined using the Stewart assay . Brieflyμm pore size, polycarbonate filters, MF-Millipore, Microglass Heim, Italy) and analyzed with a detector at 90° angle. For measuring the particle size distribution, polydispersity index (PI) was used. For each formulation, the mean diameter and PI were calculated as the mean of three different batches. The average diameter and the size distribution of each formulation were determined. The results were expressed as liposome mean diameter (nm) and polydispersity index (PI). The zeta potential (ζ) of liposomes was performed by the Zetasizer Nano Z . Briefly, an aliquot of each sample (10 μL) was diluted in filtered water and analyzed. The results were calculated by the average of 20 measurements.Dimensional analysis was performed at 20°C by photon correlation spectroscopy (PCS) . Each sample was diluted in deionizer/filtered water  equipped with a security guard. The mobile phase was a mixture 40 : 60 (v/v) of acetonitrile and filtered water. The analyses were performed in isocratic condition, at a flow rate of the mobile phase of 1 mL/min and at room temperature. The amount of UT-II, present on the surface of nanocarriers, was determined as follows. Briefly, 1 mL of conjugated liposomes dispersion was ultracentrifugated  at 80,000 rpm, at 4°C for 40 min. Supernatants were carefully removed, and the amount of UT-II unlinked to liposomes was determined by RP-HPLC. The amount of UT-II was calculated by means of a calibration curve with UT-II samples. The results have been expressed as follows:actual is the UT-II found in the supernatant and UT-IItheor is the theoretical UT-II added to the liposomes.The amount of UT-II linked to the liposomes was carried out by a high-performance liquid chromatography (HPLC) method. The HPLC system consisted of an isocratic pump  equipped with a 7725i injection valve  and SPV-10A UV-Vis detector (Shimadzu) set at the wavelength of 254 nm. The system was controlled by a SCL-10A VP System Controller (Shimadzu) connected to a computer. Chromatograms were acquired and analyzed by a Class VP Client/Server 7.2.1 program . The quantitative analysis of UT-II was performed by reverse-phase chromatography (RP-HPLC) on a Luna 5 http://www.atcc.org). PC3, DU145, and WIDR cell lines were grown in DMEM, while LNCaP and LoVo were grown in RPMI 1640. Both cell media were supplemented with 10% heat-inactivated fetal bovine serum, 20 mM HEPES, 100 U/mL penicillin, 100 mg/mL streptomycin, 1% L-glutamine, and 1% sodium pyruvate. All the cells were cultured at a constant temperature of 37°C in a humidified atmosphere of 5% carbon dioxide (CO2).Prostate  and colon (WIDR and LoVo) cancer cell lines were obtained from American Type Culture Collection  was added (20 μL/well), and the plates were incubated for further 4 h at 37°C. The MTT-formazan crystals were dissolved in 1N isopropanol/hydrochloric acid 10% solution. The absorbance values of the solution in each well were measured at 570 nm using a BioRad 550 microplate reader . Percentage of cell viability was calculated asCell proliferation was evaluated by MTT assay. Briefly, cells were seeded in serum-containing media in 96-well plates at the density of 2 × 1050) were obtained, and these values were used for subsequent experiments. MTT assay was carried out by triplicate determination on at least three separate experiments. All data are expressed as mean ± SD.Then, the concentrations inhibiting 50% of cell growth (ICUntreated cells were fixed for 20 min with a 3% PFA solution and permeabilized for 10 min with 0.1% (v/v) Triton X-100 in phosphate buffered saline at room temperature. To prevent nonspecific interactions of antibodies, cells were treated for 2 h in 5% (w/v) BSA in phosphate-buffered saline and then were incubated with a specific rabbit Ab raised against UTR  for 2 h at 37°C. After several washes, cells were incubated with a secondary goat anti-rabbit IgG-FITC  diluted at 1 : 50 in blocking solution for 1 h at room temperature. The FITC fluorescence was measured with a FACSCalibur flow cytometer (Becton Dickinson) on 10,000 cells for each sample using the CellQuest software (Becton Dickinson). Mean fluorescence intensity (MFI) of anti-rabbit-FITC sample was considered as 100%.α-tubulin were purchased from Santa Cruz Biotechnology. Blots were then developed using enhanced chemiluminescence detection reagents ECL  and exposed to X-ray film. All films were scanned by using Quantity One software (BioRad Chemi Doc).Equal amounts of cell proteins from untreated cells were separated by SDS-PAGE electrophoresis . Proteins were electrotransferred to nitrocellulose membrane by using Trans Blot Turbo . Membrane were washed in TBST  and were incubated with specific MAbs and later with horseradish peroxidase-conjugated secondary antibodies. The rabbit antibodies raised against UTR and the mouse antibody raised against μM) of Bodipy-labeled liposomes, DU145 and PC3 cells were fixed for 20 min with a 3% PFA solution and permeabilized for 10 min with 0.1% (v/v) Triton X-100in phosphate-buffered saline at room temperature. To prevent nonspecific interactions of antibodies, the cells were treated for 2 h in 5% (w/v) BSA in phosphate-buffered saline and then were incubated with a specific mouse Ab raised against vimentin  for 2 h at 37°C. After several washes, the cells were incubated with a secondary IgG goat anti-mouse-Alexa Fluor 633  diluted at 1 : 50 in blocking solution for 1 h at room temperature. The slides were mounted on microscope slides by Mowiol. The analyses were performed with a Zeiss LSM 510 microscope equipped with a plan-apochromat objective X 63 (NA 1.4) in oil immersion. The fluorescence of the Bodipy-labeled liposomes and vimentin was collected in the multitrack mode using BP550-625 and LP650 as emission filters, respectively.BD FACSCalibur fluorescent-activated flow cytometer and the BD CellQuest software (BD Biosciences) were used to perform flow cytometry analysis of liposome intracellular distribution. For these experiments, Bodipy-labeled liposomes were used on DU145 and PC3 cells. Bodipy is a bright, green-fluorescent dye with similar excitation and emission to fluorescein (FITC) or Alexa Fluor 488 dye. Green fluorescent-labeled liposomes were added to the medium of DU145 or PC3 cells at a fixed concentration. After 6, 24, 48, and 72 h of incubation at 37°C, removal of unattached liposomes was accomplished by washing the cells with phosphate-buffered saline at pH 7.4 for 1 min. Cells were then detached by trypsinization. The samples were subsequently washed twice, and for each sample, 10,000 cells were measured by using FACSCalibur flow cytometer (Becton Dickinson) using the CellQuest software (Becton Dickinson). After 6 h of incubation with a fixed concentration .All data were expressed as mean values ± SD of at least three independent experiments. Statistical analysis was obtained using ANOVA, and significant differences were determined at 2-terminal of the peptide to provide a unique site for the conjugation with the nanocarrier. The conjugation has been carried out at the extremity of PEG chain present on the liposome surface by using maleimide groups that react with cysteine residues on ligands to form stable thioether bonds. To this aim, liposomes with DSPE-PEG2000-maleimide were used for the conjugation. For comparison purpose, liposomes with DSPE-PEG2000 were also prepared. The characteristics of LipoUT, i.e., mean diameter, PI, and zeta potential, are summarized in ζ of liposomes, suggesting that negligible amount of the drug was associated with the liposome surface following purification. The different surfaces of Lipo and LipoUT, due to the presence of DSPE-PEG2000 and DSPE-PEG2000-maleimide, respectively, had only a slight influence on the doxo encapsulation process.Here, we used a short peptide of eight amino acids (peptide 4-11) of the UT-II with only one binding site. In particular, a cysteine was added to the NHIn order to assess the ability of UT-II-conjugated liposomes to efficiently deliver doxo in cancer cells that express UTR, we evaluated the expression of the receptor in different human prostate  and colon (WIDR and LoVo) cancer cell lines.The UTR expression was investigated by western blotting in colon cancer cells. In detail, both cell lines expressed the receptor, but the level of expression was higher in WIDR (about 4-fold) compared to LoVo . FACS anIn conclusion, UTR protein was expressed in all the assessed cell lines; the level of expression was higher in WIDR, PC3, and LNCaP cells compared with LoVo and DU145.To investigate if UT-II peptide conjugation to liposomes can enhance uptake and improve intracellular distribution of the formulations, FACS and confocal laser scanning microscopy (CLSM) were performed on DU145 and PC3 cells treated with fluorescent-labeled liposomes. The fluorescence associated to the cells was evaluated by FACS after 6, 24, 48, and 72 h of incubation with Bodipy-labeled liposomes, used at a fixed concentration.In particular, in both cell lines (DU145 and PC3), after 6 h of incubation with LipoUT or Lipo, FACS analysis detected the % of mean fluorescence intensity (MFI) as compared with the untreated controls . MFI of The intracellular distribution of Bodipy-labeled liposomes after 6 h of incubation was studied by confocal microscopy analysis. CLSM showed a higher widespread and intense green fluorescence intensity, both in DU145 and PC3 cells incubated with UT-II-conjugated liposomes (LipoUT) compared to Lipo Figures . MoreoveThe effects of free doxo, Lipo and liposome encapsulating doxo, and conjugated (LipoUT-doxo) or not (Lipo-doxo) with UT-II were evaluated on the proliferation of prostate  and colon (WIDR and LoVo) cancer cell lines by MTT assay as reported in “Materials and Methods.” Doxo, Lipo-doxo, and LipoUT-doxo induced a dose-dependent growth inhibition in all the cell lines after 72 h, while treatment with Lipo did not produce significant cytotoxic effects  after 72 h of treatment. After 72 h, IC50 of free doxo were equal to 0.6 μM, 4 μM, 1 μM, 0.6 μM, and 1.25 μM for DU145, PC3, LNCaP, WIDR, and LoVo, respectively  increased cell proliferation, in particular, in PC3, LNCaP, and WIDR that overexpress UTR. On these bases, we tested all the formulations by using a very high concentration of doxo, to antagonize the effect of UT-II on cell proliferation , versatility of the formulation, possibility to encapsulate drugs with different lipophilicity, design relatively easy, and possible scale-up for large production in GMP quality. Liposomes have been extensively used in cancer therapy to change drug biodistribution. In particular, liposomes allow addressing the encapsulated drug towards tissues characterized by vessels with an enhanced permeability of the endothelium, such as tumors , 30. SteHere, stealth liposomes were proposed as systems to target cancer cells overexpressing UTR and to promote drug delivery of anticancer agents, i.e., doxorubicin, into colon and prostate cancer cells. UT-II is a vasoactive peptide able to bind the UTR, a receptor involved in a number of physiological processes in different organs. This receptor has been found to be involved in proliferation, motility, and invasion of several cancer cells; among them are the colon  and humaThe conjugation of ligands able to specifically bind receptors that are overexpressed on cancer cells to liposome surface represents an efficient active targeting strategy to enhance selectivity and efficiency of drug delivery systems. This is especially important to reduce the toxic effect of the antitumor therapies. Thus, specific ligands  able to bind receptors overexpressed on the surface of cancer cells have been coupled on the liposome surface.2-terminal of the peptide to provide a unique site for the conjugation with the nanocarrier. The introduction of the cysteine residue was followed by purification and characterization , thus excluding the presence of by-products, i.e., products derived by the reduction of the disulfide bridge. Moreover, the formulation proposed in this study has been developed following an optimization study to find the experimental conditions for the highest liposome/UT-II conjugation degree. In our case, a conjugation degree of about 61% was achieved (checked by HPLC). It is worthy of note that a significant percentage of the maleimide residues are not exposed on the external surface of the liposomes, thus suggesting that the percentage of conjugation obtained here is close to the highest possible. Then, the obtained nanocarriers were loaded with a cytotoxic agent, doxorubicin (doxo), widely used in the clinical setting. Doxorubicin (doxo) is an anthracycline with significant anticancer properties. Unfortunately, its use is limited by the induction of severe cardiotoxicity that occurs through poorly investigated mechanisms. Recently, it was found [2+) and consequently activate Ca2+-dependent kinases (CaMKs) leading to cell proliferation [It is worthy of note that when considering proteins, such as transferrin or monoclonal antibodies, different binding sites are present on each molecules. Therefore, their conjugation with nanovectors can lead to a heterogeneous conjugate with a quality of the final product difficult to control. Based on these considerations, we used a short peptide of eight amino acids (peptide 4-11) of the UT-II with only one binding site. In particular, a cysteine was added to the NHas found  that doxferation . In thisferation .remote-loading method [ζ of liposomes, suggesting that negligible amount of the drug was associated to the liposome surface following purification. On the other hand, the conjugation of UT-II to liposomes did not significantly affect the mean diameter of the liposomes when compared to Lipo-doxo but produced a PI increase and a significant zeta potential decrease. Despite this, liposomes with a mean diameter of about 169 nm can still be considered suitable for intravenous administration, especially when considering the PI of about 0.2. Moreover, after conjugation of UT-II with liposomes, LipoUT compared with the unconjugated liposomes showed different ζ, due to the presence of peptide linked on the PEG extremity. In fact, the percentage of UT-II, present on the surface of liposomes, was about 61%.Doxo was also chosen because it can be easily encapsulated into liposomes by the well-known g method . This teg method . Thus, ag method . Similarg method . The encIn order to assess the ability of UT-II-conjugated liposomes to efficiently deliver doxo in cancer cells that express UTR, we evaluated the expression of the receptor in different human prostate  and colon (WIDR and LoVo) cancer cell lines.UTR protein was expressed in all the assessed cell lines; the level of expression was higher in WIDR, PC3, and LNCaP cells compared with LoVo and DU145. To investigate if the UT-II peptide conjugation to liposomes can enhance uptake and improve intracellular distribution of the formulations, FACS and confocal laser scanning microscopy (CLSM) were performed on DU145 and PC3 cells treated with fluorescent labeled liposomes. In particular, we found that cell uptake of Bodipy-labeled liposomes in PC3 and DU145 was higher for LipoUT than the not armed counterparts but at major extent in PC3 cells that express higher UTR levels.Many efforts have been made to increase selectivity and the efficacy of the liposome delivery. The strategy of active targeting has been successfully used to specifically deliver drugs to cancer cells. Moreover, it is possible to target receptors able to be internalized upon binding with the ligands, thus promoting the intracellular delivery of the drug associated with the ligand, with consequent increase of the cytotoxicity , 15. UTR50. These findings seem in contrast with observation of confocal microscopy in which higher liposome uptake was observed in UTR overexpressing cells. However, it is worthy of note that the activation of UTR by the agonist UT-II can induce cell proliferation as described in our previous work [On these bases, the effects of free doxo, Lipo, LipoUT-doxo, and Lipo-doxo were evaluated on the proliferation of prostate  and colon (WIDR and LoVo) cancer cell lines. LipoUT-doxo was more active than Lipo-doxo on the growth inhibition of cells that overexpressed UTR (LNCaP and WIDR), while in LoVo and DU145 cell lines, the activity was similar to or lower than that one of Lipo-doxo, respectively. Although PC3 express high levels of UTR, LipoUT-doxo resulted to be less cytotoxic than Lipo-doxo based on the evaluation of ICDifferent anticancer drugs have been incorporated in nanocarriers to address several issues, such as the poor solubility of drugs, the drug instability in biological fluids, and serious side effects due to the drug accumulation into the healthy tissues together with low concentration at the action site . More reIn conclusion, we have developed liposomes conjugated with UT-II (LipoUT) for efficient targeting of cancer cells that overexpress UTR and promoting drug delivery of an anticancer agent, i.e., doxorubicin, into colon and prostate cancer cells. We demonstrated that LipoUT-doxo was more active than Lipo-doxo on the growth inhibition of cells that overexpressed UTR . Moreover, we found that cell uptake of Bodipy-labeled liposomes in PC3 and DU145 was higher for LipoUT. Taken together, the data obtained in this work suggest that the encapsulation of doxo in UT-II-targeted liposomes potentiated its delivery in UTR overexpressing cells and could represent a new tool for the targeting of prostate and colon cancer.


P. americana on multidrug resistance of BEL-7402/5-FU cells, as well as a preliminary investigation on their mechanism of action. A methylthiazolyl tetrazolium (MTT) method was applied to determine the multidrug resistance of BEL-7402/5-FU, while an intracellular drug accumulation assay was used to evaluate the effects of a column chromatography extract (PACC) and defatted extract (PADF) from P. americana on reversing multi-drug resistance. BEL-7402/5-FU reflected high resistance to 5-FU; PACC and PADF could promote drug accumulation in BEL-7402/5-FU cells, among which PADF was more effective than PACC. Moreover, results from the immunocytochemical method showed that PACC and PADF could downregulate the expression of drug resistance-associated proteins ; PACC and PADF had no effects on the expression of multidrug resistance-associated enzymes (GST-π), but PACC could increase the expression of multidrug resistance-associated enzymes (PKC). Results of real-time fluorescence quantitative PCR revealed that PACC and PADF were able to markedly inhibit the expression of multidrug resistance-associated genes ; PACC presented a significant impact on the gene expression of multidrug resistance-associated enzymes, which increased the gene expression of GST-π and PKC. However, PADF had little impact on the expression of multidrug resistance-associated enzymes. These results demonstrated that PACC and PADF extracted from P. americana could effectively reverse MDR in BEL-7402/5-FU cells, whose mechanism was to inhibit the expression of P-gp, MRP, and LRP, and that PADF was more effective in the reversal of MDR than did PACC. In addition, some of extracts from P. americana altered (sometimes increasing) the expression of multidrug resistance-associated enzymes.The present study reports the reversing effects of extracts from  Hepatocellular carcinoma (HCC), which has the features of high degradation, high recurrence, and metastasis rates and whose mortality ranks third among global cancers, is the seventh most common malignant tumor type ,2. ThereP. americana (Blattodea: Blattidae), commonly known as “cockroaches”, is the medicine material of three single TCMs for the treatment of wound-healing, hepatitis, and heart failure in China [Sheng Nong’s Herbal Classic [Compendium of Materia Medica. Though P. americana is also applied as an anti-tumor remedy by local minorities in Southwestern China, studies on its efficacy in reversing MDR are rare.Researchers have disclosed that some active components found in traditional Chinese medicines (TCMs), such as psoralen, matrine, tetrandrine, and elemene, were related to the reversion of MDR, whose mechanisms of action have required further exploration . Insectsin China . The med Classic  and CompP. americana in the HCC MDR cell line of BEL-7402/5-FU and to explore their mechanisms of action by measuring the expression of multidrug resistance-associated proteins and genes.Therefore, the aim of the present study was to investigate the reversing effects of extracts from As shown in 50 , and protein kinase C (PKC); (3) MDR mediated by apoptosis-associated genes [The mechanisms of MDR are very complex, and the main molecule-biological mechanisms of MDR in tumor cells were as follows: (1) drug efflux mediated by transport proteins ,15, suched genes , such ased genes . Among ted genes ,20.P. americana can reverse multidrug resistance in tumors. Results also indicated that PACC and PADF extracted from P. americana can promote ADM accumulation in BEL-7402/5-FU cells, whose mechanism is to reduce the efflux of chemotherapeutic drugs to reverse MDR.In this study, BEL-7402 cells and BEL-7402/5-FU cells were selected as a counterpart pair of drug sensitive and drug-resistant cells. From the results of the cell growth curve, we know that the growth cycle of BEL-7402/5-FU cells, compared with that of BEL-7402 cells, is significantly prolonged. Sensitivity to chemotherapeutic drugs, which provides viable and practical conditions for the intracellular accumulation of the drug assay, indicates that BEL-7402/5-FU cells have strong resistance to 5-FU and perform cross-resistance to ADM. The intracellular accumulation assay is used to confirm whether the PACC and PADF extracted from The protein and gene expressions of P-gp, MRP, LRP, GST-π, and PKC in BEL-7402/5-FU cells were analyzed by an immunocytochemical method and real-time fluorescence quantitative PCR. It can be inferred that PACC and PADF reverse MDR, likely by downregulating the protein and gene expressions of P-gp, MRP, and LRP to inhibit the protein function and decrease drug efflux from cells. However, it is difficult to profoundly explore the effects and mechanisms because the multidrug resistance-associated enzyme system is complex. On the other hand, the observed were most statistically significant, possibly because the tested extracts (PACC and PADF) contained a mixture of several compounds. Thus, PACC and PADF should be separated and purified for further study. Meanwhile, although both PACC and PADF could reverse MDR to some degree, the effects of PADF on reversing MDR are better and stronger than PACC under non-toxic doses for cells, and the extraction process of PACC is more complicated. Consequently, PADF, being more effective and easier to extract, might be further studied in order to isolate compounds to reverse MDR phenotype of cancer cells.To date, there are two measures for reversing MDR in the clinical treatments of tumors: (1) the use of chemotherapeutic drugs, which, when combined with reversal agents, resist MDR, and (2) the use of drugs to which patients are sensitive, or an increased dosage. The latter method is widely used, but the effect is not ideal. Although the present study shows that PACC and PADF can reverse MDR in BEL-7402/5-FU cells, the mechanism here clarified could only be justified by the protein and gene expressions of P-gp, MRP, and LRP; thus, further studies about drug resistance-associated enzyme systems are still needed to provide an experimental and theoretical basis to overcome MDR and to improve the efficacy of the combination of PACC and PADF with chemotherapeutic drugs in HCC cells. In addition, mechanisms of MDR are very complex in tumors as a result of the combined action of multiple genes and factors. In the future, further studies attempting to verify whether there are other mechanisms of PACC and PADF in reversing MDR should be continued. At the same time, in vivo experiments to confirm the reversal effects of PACC and PADF are also necessary.P. americana, PACC and PADF, were prepared and kindly provided by Dr. Cheng-Gui Zhang and his coworkers at the National–Local Joint Engineering Research Center of Entomoceutics, Dali University .Human hepatocellular carcinoma BEL-7402 cells and multi-drug resistant human hepatocellular carcinoma cell line BEL-7402/5-FU cells were obtained from Shanghai Meixuan Biological Technology Co., Ltd. . Bioactive extracts of P. americana polypides, after the drying process, were crushed and cold-soak-extracted three times with 70% alcohol. Each time, the extracted liquid was filtered, and the filtered fluid was merged together and concentrated. Ultimately, an extract was prepared for the experiments [The extraction method of PADF (fulvous and viscous cream) is as follows: the eriments .The extraction method of PACC (freeze-dried powder) is as follows: the resulting extract above was added to a polyamide column and eluted with water and different proportions of aqueous methanol, collected quantitatively, and concentrated separately. One stroke segment after freeze-drying was used as a sample for the experiments .RPMI-1640 medium was bought from Hyclone  and fetal bovine serum from Gibco ; a mixture of penicillin and streptomycin, 0.25% Trypsin-EDTA, dimethyl sulfoxide (DMSO), and methylthiazolyl tetrazolium (MTT) were all obtained from Solarbio Co.  5-FU was bought from Tianjin Kingyork Amino Acid Co. ; adriamycin (ADM) was bought from Shanxi Pude Pharmaceutical Co. ; cispaltin (DDP) was bought from Qilu Pharmaceutical Co.  Fluorescence quantitative PCR kits were provided by Shanghai Solmon Biotechnology Co. , Ltd, and primers were achieved from Shanghai Sangon Biotechnology Co. . immunohistochemical staining kits were provided from Wuhan Boster Biological Co. .2 incubator, then the medium was changed every 2 days and the next generation was produced after culturing for 3–4 days [BEL-7402 cells were regularly cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37 °C in 5% CO3–4 days . BEL-7403–4 days . Experim5 cells per milliliter (with 100 μL cell suspension and 900 μL medium) for 24 h. Every 24 h, three wells were randomly selected and digested to prepare single-cell suspension for cell counting and a drawing growth curve. The Patterson method [BEL-7402 cells and BEL-7402/5-FU cells of logarithmic growth phase were digested by 0.25% Trypsin-EDTA and seeded in a 24-well plate at 1 × 10n method  was appl4 cells per well with 100 μL for culturing 12 h. A range of concentrations of 5-FU, DDP, and ADM  were added to designated wells with 100 μL per well. Meanwhile, the blank control group only contained a medium and a negative control group without drugs, which only contained cells that were set for experiments. Each concentration was repeated in 6 wells. After 48 h, the cells were incubated for 4 h following the addition of 20 μL of 5 mg/mL 3--2,5-diphenyltetrazolium bromide (MTT), the old medium was then abandoned, and 150 μL of DMSO was added to each well to measure an absorbance value at 490 nm [BEL-7402 cells and BEL-7402/5-FU cells in logarithmic growth phase were digested with 0.25% Trypsin-EDTA and seeded in a 96-well plate at 2 × 104 cells per well with 100 μL for 12 h. A range of concentrations of PACC and PADF  were added to designated wells, with 100 μL per well. Meanwhile, the blank control group only contained medium and a negative control group without drugs, which also only contained cells that were set for experiments. Each concentration was repeated in 6 wells. After 48 h, the absorbance value per well was measured at 490 nm by the aforementioned MTT method, and the IR was calculated by the same formula to evaluate IC5 and IC10 value for setting the nontoxic doses for reversal experiments. IC5 and IC10 can be obtained from IR of different concentrations by Spss 17.0.BEL-7402/5-FU cells of logarithmic growth phase were digested by 0.25% Trypsin-EDTA and seeded in a 96-well plate at 2 × 10Group 1: BEL-7402 cells. Group 2: BEL-7402/5-FU cells. Group 3: BEL-7402/5-FU cells treated with 50 μg/mL PACC. Group 4: BEL-7402/5-FU cells treated with 85 μg/mL PACC. Group 5: BEL-7402/5-FU cells treated with 23 μg/mL PADF. Group 6: BEL-7402/5-FU cells treated with 45 μg/mL PADF.4 cells per well for 12 h incubation. After 12 h, PACC and PADF were added to designated wells with 100 per well and incubated at 37 °C in 5% CO2. Drugs were added into designated wells according to the experimental groups. After 48 h, ADM was added in cells for treating 2 h. After that, cells were washed three times by cold PBS and fixed for 30 min with 4% paraformaldehyde (PFA) away from light. Then round coverslip was washed by PBS two times, stained for about 3–5 min with 1–2 drops of diamidinophenylindole (DAPI) and mounted by a mounting solution, and then observed via laser scanning confocal microscopy (LSCM).BEL-7402/5-FU cells were digested and seeded in slides to prepare a round coverslip at 5 × 104 cells per well with for 12 h incubation. PACC and PADF were added to designated wells with 100 per well and incubated at 37 °C in 5% CO2. Drugs were added into designated wells according to the experimental groups. Drugs were added into designated wells according to the experimental design. After 48 h, the immunocytochemical assay was used to detect multidrug resistance-associated proteins according to the operating instructions of the kits.Immunocytochemical assay was used to observe the effects of PACC and PADF on multidrug resistance-associated proteins in BEL-7402/5-FU cells. BEL-7402/5-FU cells were digested and seeded in slides to prepare round coverslip at 5 × 105 cells per milliliter with 2 mL per well for 12 h incubation. PACC and PADF were added to designated wells at 100 μL per well based on the former experimental groups.A real-time fluorescence quantitative PCR assay was used to confirm the expression of multidrug resistance-associated genes and enzymes and to observe the effects of PACC and PADF on the expression of multidrug resistance-associated genes and enzymes. BEL-7402/5-FU cells were digested and seeded in a 12-well plate at 1 × 10Drugs were added into designated wells according to the experimental design. After 48 h, RNA was extracted with a Trizol one-step method and dissolved by diethyl pyrocarbonate (DEPC). The concentration of RNA was determined with a spectrophotometer to establish a 10 μL reaction system of reverse transcription based on the operating instructions of the fluorescence quantitative PCR kits with the following conditions: pre-degenerated at 95 °C for 5 min, degenerated at 95 °C for 30 s, renaturated at 58 °C for 30 s, and extended at 72 °C for 30 s with 40 cycles. β-actin was used as an internal control for the loading. The forward and reverse PCR primers were as follows .t minus N0; N0 is the number of initial cells; Nt is the number of terminal cells;Doubling time (DT) was calculated according to the following formula (1):x is the absorbance value of the treatment group, and A0 is the absorbance value of the control group.The inhibitory rate (IR) was calculated according to the following formula (2):50(R) is the IC50 value of drug-resistant cell BEL-7402/5-FU, and IC50(S) is the IC50 value of sensitive cell BEL-7402. All experiments were repeated three times independently. All experiments were repeated three times and took the average value.Resistance index (RI) was calculated according to the following formula (3):50 was obtained from the following formula (4):m is the largest concentration, I is dilution factor, P is sum of inhibitory rate, Pm is the maximum IR, Pn is the minimum IR.IC(tar) is the Ct value of the target gene, while Ct(hou) is the Ct value of the housekeeping gene, β-actin.The relative expression quantity (RQ) of MDR1, MRP1, and LRP were calculated by the following formula (5):p-value of less than 0.05 was considered statistically significant.Data were expressed by mean ± SD. One-way analysis of variance (ANOVA) analysis was used to determine the differences among the study groups with SPSS v.19.0.  A P. americana altered (sometimes increasing) the expression of multidrug resistance-associated enzymes.In conclusion, the present study demonstrated that PACC and PADF could promote ADM accumulation in BEL-7402/5-FU cells so that the cross-resistance could be reduced to some extent, which was significant for achieving a reversal of MDR. PACC and PADF could inhibit the expression of multidrug resistance-associated proteins  in BEL-7402/5-FU cells and also downregulate the expression of multidrug resistance-associated genes . These results implied that PACC and PADF reverse MDR of HCC by inhibiting the expression of multidrug resistance-associated proteins and genes. Moreover, PADF was more effective in the reversal of MDR than PACC. In addition, some of extracts from


Staphylococcus aureusΔmvaS mutant can revert to prototrophy. This mutant couldn’t grow in the absence of MVA. However, after a long lag-phase of 4–6 days the mutant adapted from auxotrophic to prototrophic phenotype. During that time, it acquired two point mutations: One mutation in the coding region of the regulator gene spx, which resulted in an amino acid exchange that decreased Spx function. The other mutation in the upstream-element within the core-promoter of the mevalonolactone lactonase gene drp35. This mutation led to an increased expression of drp35. In repeated experiments the mutations always occurred in spx and drp35 and in the same order. The first detectable mutation appeared in spx and allowed slight growth; the second mutation, in drp35, increased growth further. Phenotypical characterizations of the mutant showed that small amounts of the lipid-carrier undecaprenol are synthesized, despite the lack of mvaS. The growth of the adapted clone, ΔmvaSad, indicates that the mutations reawake a rescue bypass. We think that this bypass enters the MVA pathway at the stage of MVA, because blocking the pathway downstream of MVA led to growth arrest of the mutant. In addition, the lactonase Drp35 is able to convert mevalonolactone to MVA. Summarized, we describe here a mutation-based two-step adaptation process that allows resuscitation of growth of the ΔmvaS mutant.In this study we addressed the question how a mevalonate (MVA)-auxotrophic  The very large and diverse class of isoprenoids is composed of 1000s of different organic compounds. Representatives can be found in all living organisms, where they fulfill essential roles. They are involved in the electron transport during aerobic respiration (quinones), the cell wall synthesis (bactoprenols), the photosynthesis (carotenoids), membrane stabilization, protein translation and degradation, gene transcription and many more . All isoStaphylococcus species that use the MVA pathway are often associated with humans and primates, whereas the MEP pathway is predominantly found in species linked to companion animals, livestock, and wildlife  catalyzed by the HMG-CoA synthase (MvaS)  . HMG-CoAe (MvaS) . This pae (MvaS)  and the e (MvaS) . The reaBecause of the importance of isoprenoids during different cellular processes the biosynthesis of IPP is essential for all living organisms.S. aureus, for example, repressing the expression of each of the MVA pathway genes drastically reduced growth, and a temperature-sensitive mvaA mutant was unable to grow at high temperatures (In eratures .ΔmvaS) in S. aureus and could show that this mutant was unable to grow in the absence of MVA in the medium, meaning that the mutant was auxotrophic for MVA  casein peptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 0.1% (w/v) K2HPO4, 0.1% (w/v) glucose, pH 7.2], S. aureus cells were grown at 37°C in tryptic soy broth  or BM. E. coli cultures were supplemented with ampicillin (100 μg/ml), when appropriate. In case of S. aureus chloramphenicol  or tetracycline  was added.All the bacterial strains used in this study are listed in Supplementary Table us HG001  was usedli DC10B  was usedli DC10B , pPTtuf li DC10B  and pRABli DC10B . Their dli DC10B . E. colic-41tdrp35, T11Ispx and c-41tdrp35/T11Ispx) and knockout mutants  in HG001 and ΔmvaS was performed using the plasmid pBASE6 and allelic replacement as described in  and ligating it into the BglII-site of pRAB11 or the EcoRI-site of pPTtuf. The resulting plasmids pBASE_drp35-KO, pBASE_spx-KO, pBASE_mvaA-KO, pBASE_c-41tdrp35-KI, pBASE_T11Ispx-KI, pPTtuf_drp35-strep and pRAB11_spxDD were transformed into the appropriate strains by electroporation.Oligonucleotides and plasmids are listed in Supplementary Tables ribed in . BrieflymvaS mutants (ΔmvaSad) were selected as described before -MVL (Sigma–Aldrich), and grown overnight. Afterward, ΔmvaS was inoculated into fresh TSB without MVL to an OD578 of 0.005 to keep the influence of residual MVL as low as possible. This culture was incubated for several days at 37°C under aerobic conditions and the OD578 was monitored every 24 h. As soon as the culture reached the stationary growth phase a fresh culture was inoculated, and it was streaked on tryptic soy agar (TSA) plates to get single colonies. After several days of incubation one of the biggest colonies was taken, streaked again and used for all upcoming experiments.Adapted d before  with somΔmvaS knock-in mutants were done by incubating the cells in TSB, supplemented with 500 μM (±)-MVL (TSBMV A), overnight. Cultures were then diluted to an OD578 of 0.01 and further serially diluted 1:10. Finally, 10 μl of each dilution was dropped on a TSA plate and incubated at 37°C for 5 days.Growth experiments with the TM FL Vancomycin  for cell wall staining and FM5-95  for membrane staining for 10 min at 37°C. To remove unbound dye cells were washed twice in PBS and finally resuspended in PBS. For fluorescence microscopy, bacteria were mounted on microscope slides covered with a thin film of 2% agarose dissolved in PBS. Fluorescence micrographs were obtained using a Zeiss Axio Observer Z1 LSM 800 equipped with Airyscan detector and C Plan-Apo 63x/1.4 Oil DIC objective (Zeiss). Image acquisition and analysis were performed via ZEN 2.3 image analysis software package (Zeiss).Cells were grown to the mid-exponential growth phase and washed with PBS. The pellets were then resuspended in PBS and incubated with BODIPYmvaSad mutant during mid- and late exponential growth phase using the acid guanidinium thiocyanate-phenol-chloroform extraction method described in RNA for microarray was isolated from bacterial cultures of HG001 and its Δin situ synthesis of 60-base-long oligonucleotide probes , selected as previously described  following the manufacturer’s instructions. Labeled products were then purified onto QiaQuick columns (Qiagen). Purified genomic DNA from the different sequenced strains used for the design of the microarray was extracted , labeled with Cy5 dCTP using the Klenow fragment of DNA polymerase I , and used for the normalization process  and an arbitrary cutoff of twofold for expression ratios.Fluorescence intensities were extracted using Feature Extraction software . Local background-subtracted signals were corrected for unequal dye incorporation or unequal load of the labeled product. The algorithm consisted of a rank consistency filter and a curve fit using the default LOWESS  method. Data consisting of three independent biological experiments were expressed as log 10 ratios and analyzed using GeneSpring, version 8.0 . A filter was applied to select oligonucleotides mapping ORFs in the HG001 genome, yielding approximately 95% coverage. Statistical significance of differentially expressed genes was calculated by analysis of variance  using Ge1 under accession number GSEXXXX for the platform design and GPL10537 for the original data set.The complete microarray data set has been posted on the Gene Expression Omnibus databaseTaq One-step RT-PCR kit . Fifty nanogram of RNA was used as template for each reaction. As positive control the housekeeping gene pykA was amplified and the OneTaq DNA Polymerase was taken for the no-RT negative control. Primers used for RT-PCR are listed in Supplementary Table Reverse Transcription-PCR (RT-PCR) experiments were carried out using the OnemvaSad as described earlier  for 24 h at 4°C while stirred after washing with water. PG was harvested by centrifugation and washed several times with water until HFA was completely removed and lyophilized. Lyophilized PG was resuspended in 25 mM sodium phosphate buffer (pH 6.8) to a final OD578 of 5.0, digested with mutanolysin overnight at 37°C, reduced with sodium borohydride and analyzed by HPLC as described earlier (Peptidoglycan was isolated from wild type HG001 and the adapted mutant Δ earlier . Briefly earlier .55-isoprenoids were extracted with methanol/chloroform/PBS as described in (55-PP) to undecaprenyl-phosphate (C55-P) (55-isoprenoids was performed by reverse-phase HPLC as described (55-PP and C55-P in one peak.Cribed in . Cells w (C55-P) . The anaescribed  with one2O2 filter disk diffusion assays were performed. Overnight cultures were diluted to an OD578 of 0.1 and streaked onto TSA with a cotton swap. Filter disks were prepared by pipetting 20 μl of diamide (50 mM) or H2O2 (10 mM) onto the disks, which were then put on the agar plates. The diameter of the zone of inhibition (ZOI) were measured after the incubation at 37°C for 18 h using the software ImageJ.To determine the sensitivity of bacterial strains to diamide, an inducer for disulfide stress, and HS. aureus HG001, harboring the plasmid pPTtuf_drp35-strep, which constitutively expresses a C-terminal strep-tagged Drp35 protein, was diluted to an OD578 of 0.1 with fresh BM and incubated for at least 4 h at 37°C. The cells were harvested by centrifugation , washed with phosphate-buffered saline (PBS), and broken down by glass beads using a FastPrep FP120 instrument . To remove cell debris the solution was centrifuged  and the supernatant was subjected to the purification procedure using Strep-Tactin Superflow resin  described by the manufacturer. During the whole procedure buffers lacking EDTA were used. After elution the purified protein was dialyzed against 20 mM Tris/HCl, pH 8.0 overnight at 4°C. Protein concentration was determined using the BCATM Protein Assay Kit (Thermo Scientific).An overnight culture of ® M200 Microplate Reader, each well contained 2 mM HEPES, pH 8.0, 1 mM CaCl2, 0.004% phenol red, 0.005% bovine serum albumin, 1 mM substrate and 5 – 10 μl purified protein. Spontaneous hydrolysis of the substrate was corrected for by substituting the enzyme by the same volume of 20 mM Tris/HCl, pH 8.0. A calibration curve with different amounts of HCl was used to calculate the rate of hydrolysis  and adjusted to a cell concentration of 5 × 108/ml in PBS. The injection volume was 10 μl, which is equal to 5 × 106 cells, per larvae. The larvae were infected with cells of S. aureus HG001 and HG001ΔmvaSad. In total 30 larvae, separated in three different experiments, were used for each strain. PBS served as a control. After injection, larvae were incubated at 37°C, and dead larvae were counted every 24 h.Larvae of eviously  with fewp < 0.05. n represents independent biological replicates.Multiple comparisons were analyzed by either ordinary one-way ANOVA or RM one-way ANOVA with Bonferroni post-test. The log-rank (Mantel-Cox) test was used to analyze the infection model. Statistical analyses were performed with GraphPad Prism software, with significance defined as mvaS gene, encoding the hydroxymethylglutaryl-coenzyme A (HMG-CoA) synthase, leads to auxotrophy for MVA; however, growth of the mutant could be restored by the addition of MVA or MVL to the medium . Surprisingly, after prolonged cultivation for about 4–6 days, the ΔmvaS mutant started to grow but reached only an OD578 of about 2.5. When samples of this culture were plated on TSA small and large colonies were observed after several days . After inoculation in fresh TSB, the cells were able to grow without a prolonged lag-phase; and when plated on TSA it formed uniformly large colonies . We named these stable variants ΔmvaSad, for adapted ΔmvaS. Although, ΔmvaSad grew without lag-phase the growth was significantly retarded and they reached only of the OD578 of the parent strain . Also, in contrast to the parent strain, colonies of ΔmvaSad were white suggesting that they were not able to produce the orange staphyloxanthin.Recently we showed that deletion of the mvaSad is due to mutations, why we performed whole genome analysis.We hypothesized that adaptation to MVA-prototrophy in ΔmvaS mutant and two independently adapted ΔmvaSad clones from different experiments were sequenced. Isolation of the chromosomal DNA and the sequencing of the genomes were performed by the DSMZ  and analyzed for single nucleotide polymorphisms (SNPs). The long-read sequencing technique PacBio RS II with an average read length of 10 kb was used (mvaSad clones two interesting SNPs could be identified (Table 1). One SNP was located in the promoter region of drp35 (SAOUHSC_03023), which encodes a lactonase-like protein with yet unknown substrate specificity and function. The ΔmvaSad 1-clone carried a base exchange from cytosine to thymine 41 nucleotides upstream of the predicted transcription start site (TSS) (c-41t). The ΔmvaSad 2-clone carried adenine instead of guanine 52 nucleotides upstream of TSS (g-52a) .The genomes of the parent strain HG001, the non-adapted Δwas used . For errmvaSad 1- and 2-clones carried a point mutation in the coding region of spx (SAOUHSC_00934), which encodes a transcriptional regulator that is involved in the global stress response  in PBP1 (penicillin binding protein 1) and in ΔmvaSad 2 a silent mutation in phoR. Since these two mutations are only present in one mutant they do not contribute to the adaptation and were therefore not considered in further experiments.Both Δresponse . The SNPresponse . In ΔmvamvaSad to MVA-prototrophy both mutated genes of ΔmvaSad 1, c-41tdrp35 and T11Ispx, were introduced separately as well as combined into the non-adapted ΔmvaS mutant. The created strains were named ΔmvaS-c-41tdrp35, ΔmvaS-T11Ispx, and ΔmvaS-c-41t/spxT11Idrp35. In a serial dilution (1:10) the clones were tested for growth on TSA in the absence of MVA . The ΔmvaS clone, which carried both mutations (ΔmvaS-c-41t/spxT11Idrp35), and ΔmvaSad grew similar. The ΔmvaS clone, that carries only T11Ispx, showed only very little growth indicated by the faint spot at the highest cell concentration. The non-adapted ΔmvaS mutant as well as ΔmvaS-c-41tdrp35 were not able to grow. This result indicates that both mutations are necessary for the adaptation of ΔmvaSad to MVA prototrophy.To confirm the importance of the two mutations in the adaptation of Δspx and drp35 is reproducible we repeated the isolation of adapted ΔmvaSad clones additional five times. In all experiments the adapted clones formed colonies with different size on TSA plates . All tested large colonies revealed SNPs in both genes, spx and drp35. The identified SNPs were located 40 to 53 nucleotides upstream of the predicted TSS of drp35, and in the codons 11 or 14 of spx, which always led to an amino acid exchange (Table 2).To investigate whether the development of the mutations in Figure 1B we also observed small colonies when the ΔmvaS mutants grew up after the long lag-phase. We wondered whether the small colonies were also mutated. Indeed, all the small colonies from five independent experiments carried a SNP in the spx gene but not in drp35 (Table 2). The results prove that the adaptation of ΔmvaS to ΔmvaSad occurs stepwise via two successive mutations, the first occurring in spx and the second in the drp35 gene.As shown in c-41tdrp35, HG001-T11Ispx and HG001-c-41t/spxT11I.drp35 In addition, a spx deletion mutant (Δspx) was constructed.For phenotypic characterizations and to elucidate the effect of the mutations on cell metabolism, SNPs were also cloned into the wild type, resulting in the strains HG001-mvaS-T11Ispx was 29-times longer compared to the parent strain (Table 3). This shows that the ΔmvaS-spxT11I mutant is able to grow but that the growth is severely impaired which is also indicated by the tiny colonies . The adapted clone ΔmvaSad grew much better, due to the additional mutation in drp35. The doubling time was “only” 8-times higher compared to the parent strain and colonies on TSA were much larger . The single mutant ΔmvaS-c-41tdrp35 was unable to grow in the absence of MVA, . This result indicates that the mutation in spx occurs first during the adaptation and that a mutation occurring first in drp35 cannot resuscitate growth.The doubling time of the single mutant, Δc-41tdrp35 did not influence growth. However, T11Ispx slightly increased the generation time 1.1-fold in both HG001-T11Ispx and HG001-c-41t/spxT11Idrp35 (Table 3). However, complete deletion of spx reduced growth 1.8-fold. The strain ΔmvaS-c-41t/spxT11Idrp35 showed a similar doubling time than ΔmvaSad. Further, both mutations had no influence on staphyloxanthin production. All knock-in strains in HG001 as well as Δspx showed the typical orange color, whereas ΔmvaSad and ΔmvaS-c-41t/spxT11Idrp35 strains were white . Fluorescence staining with BODIPYTM FL Vancomycin revealed that less vancomycin was bound in the cross wall of ΔmvaSad and spots with increased dye accumulation were visible. This suggests that less PG was present and that cross wall is largely deficient of uncross-linked PG. Membrane staining with FM5-95 revealed a weaker staining and dot-like structures . In the merged image, membrane- and PG-stained areas do not really overlap. Overall, the microscopic studies suggest that ΔmvaSad has a severe deficiency in PG synthesis and that it is osmotic fragile as indicated by the blown-up cells.Already in bright field (BF) imaging it’s visible that the cells of the adapted Δc-41tdrp35, HG001-T11Ispx and HG001-c-41t/spxT11Idrp35 showed normal cell size and cell wall staining. Only the strains ΔmvaSad and ΔmvaS-c-41t/spxT11Idrp35 showed the abnormal morphology .Because of the abnormal cell size and the irregular cell wall staining in ΔmvaSad. Undecaprenol is part of Lipid II and is needed to translocate PG precursors across the membrane. To answer this question, we extracted lipids from whole cells and the chloroform extract was analyzed for the presence of undecaprenol by RP-HPLC. We analyzed the phosphorylated undecaprenyl-phosphate (C55-P) and undecaprenyl-diphosphate (C55-PP). Chemical dephosphorylation with KOH, which converts C55-PP into C55-P, allowed the analysis and quantification of both forms in one peak. Commercial undecaprenyl-MPDA was used as a standard. The untreated standard showed one single and well-defined peak with a retention time of ∼ 30 min . The chloroform extract of the parent strain showed that most of the substances eluted during the first 5 min and that C55-P could be detected very well. The small shift of the peak of the standard can be explained by the slightly higher molecular mass (881 g/mol compared to 847 g/mol). Interestingly, the same peak could also be found in ΔmvaSad cells, but the amount was 18-fold less compared to the parent strain . The amount of phosphorylated undecaprenol in the parent strain was 282 nmol/g cell dry weight, while for ΔmvaSad it was only 16.1 nmol/g cell dry weight (Table 4). This result shows that ΔmvaSad can synthesize undecaprenol, whose synthesis normally needs IPP as precursor. Cloning of c-41tdrp35 or T11Ispx into HG001 resulted in an increase in undecaprenol to 461 and 426 nmol/g cell dry weight, respectively. The strain ΔmvaS-c-41t/spxT11Idrp35 produced similar undecaprenol amounts as ΔmvaSad.As the primary structure of PG was apparently not altered we investigated whether undecaprenol, a follow product of MVA, could be formed by Δdrp35 suggested an impact on gene expression. Also, we wondered whether the mutation in the transcriptional regulator gene spx influenced drp35-expression. In a semi-quantitative RT-PCR approach the expression of drp35 in the exponential growth phase was determined in several strains. Relative to the parent strain, drp35 was upregulated 1.7-fold in the knock-in mutant HG001-c-41tdrp35, and 1.3-fold in HG001-spxT11I . The combination of both mutations (c-41t/spxdrp35T11I) further increased drp35 expression to 1.8-fold. The deletion of the spx gene resulted in a 2.4-fold higher drp35 expression compared to the parent strain, whereas the over-expression of a proteolysis resistant Spx variant  .The mutation in the promoter of partate)  reduced mvaSad were differently expressed compared to the parent strain HG001. For that RNA was isolated from cells in the mid (t1) and late-exponential growth phase (t2). In total 181 genes were more than 2.0-fold differentially expressed at least at one of the time points. Fifty four genes were up-regulated in the adapted ΔmvaSad, whereas 127 genes were down-regulated at t1 and/or t2  (drp35 gene was upregulated 3.2- (t1) and 2.4-fold (t2), respectively.By transcriptome analysis we tried to find out which genes in Δd drp35) . Among td drp35) . The lac2O2 was investigated. Filter disk diffusion assays with diamide and H2O2 were performed and the sensitivity was compared by measuring the diameter of the ZOI. Compared to the parent strain HG001, Δspx was, as expected, much more sensitive toward both substances. Also HG001-T11Ispx was significantly more sensitive, but not as sensitive as the deletion mutant . These results, together with the results from the growth analysis and RT-PCR approach, show that the T11I-mutation in Spx reduces the activity of the regulator, but does not abolish it completely; the results also show that spx is an important regulator gene as its deletion has a severe impact on growth.Since Spx plays a key role during disulfide and oxidative stress , the senTable 5 and Supplementary Figure Table 5). For some lactones we didn’t see any activity. This result suggests MVL as the physiological substrate of the lactonase Drp35.Drp35 is annotated as a lactonase and it has been shown that it can use the aromatic lactones dihydrocoumarine and 2-coumaranone as substrates . Using ac-41tdrp35 showed a 10-fold higher hydrolysis rate, and in the double mutant, HG001-c-41t/spxT11Idrp35, the activity was even 14-fold higher compared to the parent strain (Table 6). The mevalonolactonase activity of cell extracts of strain HG001-T11Ispx was slightly increased (1.6-fold) compared to HG001. Cell extracts of the adapted mutant, ΔmvaSad, and the complemented non-adapted mutant, ΔmvaS-c-41t/spxT11Idrp35, had a 18- and 10-fold, respectively, higher activity than the parent strain HG001; indicating that the mutation in c-41tdrp35 contributes more to lactonase activity than the mutation in T11Ispx.We also compared the mevalonolactonase activity in cell extracts of parent and mutant strains. Compared to the parent strain, HG001, the cell extract of strain HG001-mvaSad in the presence of FMV. FMV is an inhibitor of the diphosphomevalonate decarboxylase MvaD, which is the last enzyme in the MVAP. In the presence of FMV ΔmvaSad was not able to grow, indicating that the lower MVAP is still needed in the mutant .To investigate whether the MVAP, downstream of MvaS, is still functional, we grew the ΔmvaSad still lysed erythrocytes . However the appearance of the hemolytic zone was different. The parent strain HG001 showed only a clear halo, whereas ΔmvaSad showed in addition a turbid halo surrounding the clear halo. Wide clear zones are indicative for α-hemolysin and turbid halos for β-hemolysin activity . Larvae infected with the parent strain showed a survival rate of only 17% (5 out of 30 larvae), with more than 50% dead larvae within the first 2 days. The survival rate of larvae infected with the adapted mutant amounted to 57% (17 out of 30). In the first 2 days only 10% percent of the larvae died.The S. aureus the MVAP is the only route to synthesize the universal isoprenoid-precursor isopentenyl-pyrophosphate, which makes the MVA pathway a potential target for antimicrobial compounds. Recently, we created a ΔmvaS deletion mutant in S. aureus and, as expected, the mutant was unable to grow in medium lacking MVA . This indicates that only a few specific mutations lead to the adaptation of the mutant.We wondered whether the mutational adaptation of ΔE. coli and other bacteria were mostly gyrA mutations . Ciprofloxacin and other quinolones target the A subunit of DNA gyrase and selection of spontaneous single-step resistance mutants of utations . Resistautations . The difspx and drp35 occurred in a special order or simultaneously. A simultaneous mutation is completely unlikely as the frequency would be extremely low (multiplication of the frequency of each single mutation). So the question remained which mutation appears first or if it is a random event. When ΔmvaS started to grow after the lag-phase of 4–6 days we observed always a mixture of very small and larger colonies on agar plates . All the small colonies we investigated carried only one mutation, namely in spx. All the large colonies carried the double mutations in both spx and drp35 . We never found colonies with a mutation in drp35 only. This result indicates that the first mutation is in the regulator gene spx following the second mutation in drp35. The mutations in this order make sense, because, the T11Ispx mutation allows already slight growth, which facilitates the development of the second mutation. Growth is necessary for the second mutation, as mutations occur mainly during DNA replication  of the RNA Polymerase, a common target for transcriptional activators (spx is located in the coding region and caused an amino acid exchange from threonine to isoleucine at position 11 (T11I). The amino acid substitution is located in the so-called C-X-X-C motif, which mediates enzyme activity by the reduction of the cysteine residues. We assume that the change in this motif affects the general activity of Spx, in both positive and negative way. To see whether oxidative stress tolerance is affected, we introduced the mutation in the parent strain, HG001-T11Ispx. Indeed, this mutation made HG001-T11Ispx more sensitive against oxidative stress triggered by the thiol-reactive chemical diamide and by H2O2. Deletion of spx further increased the sensitivity, indicating, that the activity of SpxT11I is reduced but not completely abolished.Spx is described as a global transcriptional regulator in nditions . The Bactivators . The poidrp35 expression, as verified by several observations: In HG001-T11I drp35spx was 1.7-fold up-regulated and 2.4-fold in Δspx. When spx was over-expressed in HG001 (pRAB11DDspx) drp35 expression was repressed. Furthermore, the value for mevalonolactonase activity of strain HG001-T11Ispx was in between the values of Δspx and HG001. All our results suggest that the activity of SpxT11I is not abolished, but severely alters the regulator function. But why this reduced activity is beneficial for the mutant is still not completely clear.Spx is a negative regulator of c-41tdrp35 in ΔmvaSad was located at position -41, which is in the so-called UP (upstream)-element of the -35-region of the core-promoter. Comparative transcriptome analysis showed that drp35 is up-regulated two to three times in ΔmvaSad, indicating that this mutation increased drp35 expression, which could be confirmed by RT-PCR. UP-elements are in general AT-rich and stimulate gene transcription . Furthermore, when examining the location of drp35 in various staphylococcal genomes we found that in some species, like S. xylosus, drp35 is positioned directly next to mvaS and mvaA. In S. sciuri strain FDAARGOS_285 it is located directly upstream and in the same orientation of the mvaK1/D/K2 operon indicating an involvement of Drp35 in the MVA pathway (Supplementary Figure S. aureus and some other species it is dislocated. Finally, it is remarkable that Drp35 shows the highest lactonase activity with MVL.There are several reasons why we think that MVA is the bypass product. First, we investigated whether genes downstream of S. aureus with such an antibiotic would probably lead to the emergence of resistant and virulent mutants. However, in a Galleria mellonella infection model it turned out that the virulence of the adapted mutant is greatly reduced, which might be due to the missing staphyloxanthin and the more fragile cell wall. Additionally, the growth on sheep blood agar showed that c-41tdrp35 influenced hemolysin production.The upper MVA pathway, in particular MvaA, is considered as a potential target for the development of new antibiotics . TherefomvaS mutant can revert to prototrophy by acquiring two point mutations in the genes spx and drp35. The mutations led to a decreased regulator activity of Spx and to an increased expression of the lactonase Drp35. Based on these mutations the adapted mutant, ΔmvaSad, seems to be able to synthesize sufficient MVA to produce small amounts of the lipid carrier undecaprenol. However, the amount of produced MVA is not high enough to allow a fast and proper cell wall synthesis or to synthesize staphyloxanthin. The adapted ΔmvaSad mutant appears to have a rescue pathway that allows cell proliferation in the absence of the upper MVA pathway. We could not uncover this bypass completely. Nevertheless this study illustrates once again how adaptive the bacterial genome is. Like a lifeless creature a subpopulation is able to rise again by genetic adaptation. Interestingly, the adaptation includes always the two point mutations in spx and drp35 and always in the order spx first and drp35 second, a classical case of adaptive mutation.Altogether, we showed here that a MVA auxotrophic FG and SR designed the study. SR, PE, AL, MN, JS, JO, PS, PF, and FG designed the experiments. SR performed most of the experiments. E-JB and PF performed transcriptome analysis. MN performed PG analysis. BB and CS performed whole genome sequencing and analysis and PS performed fluorescence microscopy. PE contributed to proofreading. FG and SR wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer FC and handling Editor declared their shared affiliation.